Fluorescence image analyzing apparatus, image processing method of fluorescence image, and computer program

ABSTRACT

Disclosed is a fluorescence image analyzing apparatus including a light source that emits light to a sample including a plurality of cells labeled with a fluorescent dye at a target site, an imaging unit that captures a fluorescence image of each of the cells that emit fluorescence by being irradiated with the light, a processing unit that processes the fluorescence image captured by the imaging unit, and a display unit that displays the fluorescence image processed by the processing unit. The processing unit performs an extraction process of extracting, for each cell, a plurality of bright spots in the fluorescence image including the target site, a changing process of changing a pixel value of each of the plurality of extracted bright spots based on the pixel value of each bright spot, and a display process of displaying the fluorescence image whose pixel value is changed on the display unit.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority from prior Japanese Patent ApplicationNo. 2017-080851, filed on Apr. 14, 2017 entitled “FLUORESCENCE IMAGEANALYZING APPARATUS, IMAGE PROCESSING METHOD OF FLUORESCENCE IMAGE, ANDCOMPUTER PROGRAM”, the entire contents of which are incorporated hereinby reference.

TECHNICAL FIELD

The present invention relates to a fluorescence image analyzingapparatus, an image processing method of a fluorescence image, and acomputer program.

BACKGROUND

WO 2003/048300 discloses a method for treating cells when a flowcytometer or the like is applied for detection in a fluorescence in situhybridization method (FISH method). In the FISH method, first, apretreatment for hybridizing a fluorescently labeled probe to the basesequence of a target site present in the nucleus of a cell is performedto fluorescently label the target site. Subsequently, a fluorescencesignal (bright spot) generated from the fluorescently labeled probe isdetected.

In the FISH method, the fluorescence image is captured with afluorescence microscope, an imaging flow cytometer, or the like.However, even in the case where the same fluorescently labeled probe isused, the brightness may be different between bright spots in onefluorescence image due to variations in pretreatment. Also, since cellstake a three-dimensional shape in a flow cell of a flow cytometer, thebrightness of a plurality of bright spots in one captured fluorescenceimage may be different from each other depending on the position (depth)of the bright spot when light is radiated thereto, for example,depending on whether the bright spot is present on the surface of thenucleus or near the center of the nucleus. As described above, thebrightness of bright spots may vary in one fluorescence image when afluorescence image of a cell is captured in a flow cytometer. Therefore,when an operator checks the presence or absence of a bright spot in thefluorescence image captured by a flow cytometer in the FISH method,there is a case where a darker bright spot cannot be detected.

Furthermore, in a multicolor FISH method, by using a probe labeled witha green fluorescent dye and a probe labeled with a red fluorescent dye,the presence or absence of a fused bright spot in which the two probesare combined by being adjacent to each other and in which yellowfluorescence is emitted is detected. Specifically, a fused bright spotis detected by superimposing a fluorescence image obtained by imagingthe green fluorescent dye and a fluorescence image obtained by imagingthe red fluorescent dye. However, as described above, in onefluorescence image captured in a flow cytometer, the brightness of aplurality of bright spots may vary. Therefore, there is a problem that,when confirming a fused bright spot in a fluorescence image captured byusing a flow cytometer in the multicolor FISH method, for example, inthe case where a certain red bright spot is dark and a green bright spotadjacent thereto is bright, the red bright spot is overwhelmed by thegreen bright spot and the operator cannot determine that the bright spotis a fused bright spot when two fluorescence images are superimposed.

SUMMARY OF THE INVENTION

The scope of the present invention is defined solely by the appendedclaims, and is not affected to any degree by the statements within thissummary.

A first aspect of the present invention relates to a fluorescence imageanalyzing apparatus. A fluorescence image analyzing apparatus (1)according to this aspect includes a light source (120 to 123) that emitslight to a sample (10) including a plurality of cells labeled with afluorescent dye at a target site, an imaging unit (160) that captures afluorescence image of each of the cells that emit fluorescence by beingirradiated with the light, a processing unit (11) that processes thefluorescence image captured by the imaging unit (160), and a displayunit (13) that displays the fluorescence image processed by theprocessing unit (11). The processing unit (11) performs an extractionprocess of extracting, for each cell, a plurality of bright spots in thefluorescence image including the target site, a changing process ofchanging a pixel value of each of the plurality of extracted brightspots based on the pixel value of the bright spot, and a display processof displaying the fluorescence image whose pixel value has been changedon the display unit.

A second aspect of the present invention relates to an image processingmethod of a fluorescence image in which a fluorescence image of a cellobtained by measuring a sample (10) including a plurality of cells inwhich a target site is labeled with a fluorescent dye is processed. Theimage processing method according to this aspect includes an extractionstep of extracting, for each cell, a plurality of bright spots in thefluorescence image including the target site, and a changing step ofchanging a pixel value of each of the plurality of extracted brightspots based on the pixel value of the bright spot.

A third aspect of the present invention relates to computer program forcausing a computer to execute image processing of a fluorescence imageof a cell acquired by measuring a sample (10) including a plurality ofcells in which a target site is labeled with a fluorescent dye. Theimage processing of the computer program according to this aspectincludes an extraction process of extracting, for each cell, a pluralityof bright spots in the fluorescence image including the target site, anda changing process of changing a pixel value of each of the plurality ofextracted bright spots based on the pixel value of the bright spot.

According to the first to third aspects of the present invention, evenwhen a light bright spot having high pixel values and a dark bright spothaving low pixel values present in one fluorescence image of a cell fromwhich the bright spots have been extracted, the pixel values of the darkbright spot are enhanced in a changing process (changing step) such thatthe difference between the pixel values of the dark bright spot and thelight bright spot is reduced. Therefore, it becomes easier for anoperator or the like to recognize a dark bright spot by visualobservation in image observation of each fluorescence image. Inaddition, as a result of the pixel values of the dark bright spot beingenhanced in a changing process (changing step) such that the differencebetween the pixel values of the dark bright spot and the light brightspot is reduced for each fluorescence image, in the analysis of acomposite image in which a plurality of fluorescence images arecombined, a plurality of bright spots overlap with each other at a fusedbright spot in which bright spots of the fluorescence images overlapeach other, in a state in which the difference in pixel value isreduced. Therefore, determination of whether or not a spot is a fusedbright spot becomes easier, and thus it becomes easier for an operatoror the like to detect a fused bright spot by visual observation. Asdescribed above, according to the present invention, even in the casewhere a light bright spot having high pixel values and a dark brightspot having low pixel values are present in one fluorescence image,confirmation of a bright spot in image observation of each fluorescenceimage and detection of a fused bright spot in analysis of a compositeimage of a plurality of fluorescence images can be performed easily.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram showing a configuration of an embodimentof a fluorescence image analyzing apparatus;

FIG. 2A is a diagram exemplifying first to third images and a brightfield image acquired by the fluorescence image analyzing apparatus;

FIG. 2B is a schematic diagram for describing extraction of a nucleusregion performed by the fluorescence image analyzing apparatus;

FIG. 2C and FIG. 2D are schematic diagrams for describing extraction ofbright spots performed by the fluorescence image analyzing apparatus;

FIG. 3 is a flowchart for describing an operation of a processing unit;

FIGS. 4A to 4D are respectively schematic diagrams for describinginformation of bright spots of a negative pattern and positive patterns1 to 3;

FIG. 5A is a schematic diagram of a first image after an extractionprocess of first bright spots;

FIG. 5B is a schematic diagram of the first image after an emphasisprocess of the first bright spots;

FIG. 5C is a schematic diagram of the first image after a process ofchanging the pixel values of the first bright spots;

FIG. 6A is a schematic diagram of a second image after an extractionprocess of second bright spots;

FIG. 6B is a schematic diagram of the second image after an emphasisprocess of the second bright spots;

FIG. 6C is a schematic diagram of the second image after a process ofchanging the pixel values of the second bright spots;

FIG. 7A is a schematic diagram of a composite image of the first imageafter the emphasis process of the first bright spots and the secondimage after the emphasis process of the second bright spots;

FIG. 7B is a schematic diagram of a composite image of the first imageafter the process of changing the pixel values of the first bright spotsand the second image after the process of changing the pixel values ofthe second bright spots;

FIG. 8A is a schematic diagram of a composite image in which a thirdimage is combined with the composite image of the first image and thesecond image of FIG. 7A;

FIG. 8B is a schematic diagram of a composite image in which the thirdimage is combined with the composite image of the first image and thesecond image of FIG. 7B;

FIGS. 9A and 9B are diagrams for describing an example of a functionalexpression in the process of changing pixel values; and

FIG. 10 is a schematic diagram showing a configuration of anotherexample of a measurement device.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Hereinafter, embodiments of the present disclosure will be described indetail with reference to attached drawings. In the following embodiment,the present disclosure is applied to an apparatus in which a samplesubjected to a pretreatment of hybridizing a target site (targetsequence) present in the nucleus of a cell with a nucleic acid probe(hereinafter simply referred to as a probe) including a nucleic acidsequence having a sequence complementary to the target sequence andlabeled with a fluorescent dye is measured and a fluorescence imageacquired for each cell among a plurality of cells in the sample isanalyzed.

In one example of this embodiment, analysis of chromosomal abnormalitiesby a fluorescence in situ hybridization (FISH) method is performed by,for example, a flow cytometer (e.g., imaging flow cytometer), afluorescence microscope, or the like. In the following embodiment, as anexample, an embodiment in which a BCR gene on chromosome 22 and an ABLgene on chromosome 9 are set as target sites in a nucleic acid, andcells having a translocation (a BCR/ABL fusion gene, also referred to asa Philadelphia chromosome: t (9; 22) (q34.12; q11.23)) betweenchromosome 9 and chromosome 22 observed in chronic myelogenous leukemiaare measured and analyzed will be described. Chromosomal abnormalitiesdetected by the fluorescence image analyzing apparatus are not limitedas long as the abnormalities can be detected by the FISH method.Examples of chromosomal abnormalities include translocations, deletions,inversions, and duplications. Specific examples of the chromosomalabnormalities include chromosomal abnormalities associated with locisuch as BCR/ABL fusion gene and ALK gene.

In the following embodiment, cells to be measured are not limited aslong as the cells are nucleated cells. For example, the cells may benucleated cells in a specimen collected from a subject, and may bepreferably nucleated cells in a blood specimen. In this specificationand the like, the sample is a cell suspension to be subjected tomeasurement including cells derived from a specimen including a targetsite hybridized with a probe. The sample includes a plurality of cells.The number of the plurality of cells are at least 10² or more,preferably 10³ or more, more preferably 10⁴ or more, further preferably10⁵ or more, and still more preferably 10⁶ or more.

In this embodiment, an abnormal cell refers to a cell having achromosomal abnormality. Examples of abnormal cells include tumor cellssuch as cancer cells. Preferable examples of abnormal cells includehematopoietic tumor cell such as leukemia and cancer cells such as lungcancer.

FIG. 1 shows a schematic configuration of a fluorescence image analyzingapparatus 1 of this embodiment. The fluorescence image analyzingapparatus 1 shown in FIG. 1 includes a measurement device 100 and animage processing device 200 for processing a fluorescence image, andmeasures and analyzes a sample 10 prepared by a pretreatment by apretreatment device 300.

An operator collects nucleated cells that are measurement target cellsby, for example, centrifugally separating a blood specimen, collectedfrom a subject, by using a cell separation medium such as Ficoll. Incollecting the nucleated cells, the nucleated cells may be collected byhemolyzing erythrocytes or the like by using a hemolyzing agent to leavenucleated cells instead of collecting the nucleated cells bycentrifugation. The pretreatment device 300 includes a mixing containerfor mixing the nucleated cell suspension acquired by centrifugation orthe like with a reagent, a dispensing unit for dispensing the nucleatedcell suspension and reagent to the mixing container, a heating unit forheating the mixing container, and the like. The pretreatment device 300performs a pretreatment including a step of labeling a target site in acell collected from a subject with a fluorescent dye and a step ofstaining the nucleus of the cell with a nuclear dye, and thus prepares asample 10. Specifically, in the step of labeling a target site with afluorescent dye, the target sequence and a probe including a nucleicacid sequence having a sequence complementary to the target sequence andlabeled with a fluorescent dye are hybridized.

In the FISH method, a target site on a chromosome is detected by usingone or more fluorescent dyes. Preferably, in the FISH method, two ormore fluorescent dyes are used to detect a target site on a firstchromosome and a target site on a second chromosome (“first” or “second”is a concept of a comprehensive number and does not indicate achromosome number). For example, a probe that hybridizes with a BCRlocus is a nucleic acid having a sequence complementary to the basesequence of the BCR locus and is labeled with a first fluorescent dyethat generates a first fluorescence of a wavelength λ21 by beingirradiated with light of a wavelength λ11. By using this probe, the BCRlocus is labeled with the first fluorescent dye. A probe that hybridizeswith an ABL locus is a nucleic acid having a sequence complementary tothe base sequence of the ABL locus and is labeled with a secondfluorescent dye that generates a second fluorescence of a wavelength λ22by being irradiated with light of a wavelength λ12. By using this probe,the ABL locus is labeled with the second fluorescent dye. The nucleus isstained with a nuclear dye that generates a third fluorescence of awavelength of λ23 by being irradiated with light of a wavelength λ13.The light of wavelength λ11, the light of wavelength λ12, and the lightof wavelength λ13 are so-called excitation light.

More specifically, the pretreatment device 300 performs a treatment forimmobilizing cells so that the cells do not contract due to dehydration,a membrane permeation treatment of opening a hole having a size throughwhich a probe can be introduced into a cell, a heat modificationtreatment of applying heat to cells, a treatment of hybridizing thetarget site and the probe, a washing treatment of removing unnecessaryprobes from the cells, and a treatment of staining the nucleus.

The measurement device 100 includes a flow cell 110, light sources 120to 123, condenser lenses 130 to 133, dichroic mirrors 140 and 141, acondenser lens 150, an optical unit 151, a condenser lens 152, and animaging unit 160. The sample 10 is flowed through a flow channel 111 ofthe flow cell 110.

The light sources 120 to 123 irradiate the sample 10 flowing through theflow cell 110 with light. The light sources 120 to 123 are constitutedby, for example, semiconductor laser light sources. Light of wavelengthsλ11 to λ14 is respectively emitted from the light sources 120 to 123.

The condenser lenses 130 to 133 respectively collect light ofwavelengths λ11 to λ14 emitted from the light sources 120 to 123,respectively. The dichroic mirror 140 transmits light of wavelength λ11and refracts light of wavelength λ12. The dichroic mirror 141 transmitslight of wavelengths λ11 and λ12 and refracts light of wavelength λ13.In this manner, the sample 10 flowing through the flow channel 111 ofthe flow cell 110 is irradiated with the light of wavelengths λ11 toλ14. The number of semiconductor laser light sources provided in themeasurement device 100 is not limited as long as 1 or more light sourcesare provided. The number of semiconductor laser light sources can beselected from among, for example, 1, 2, 3, 4, 5 and 6.

When the sample 10 flowing through the flow cell 110 is irradiated withlight of wavelengths λ11 to λ13, fluorescence is generated from thefluorescent dye staining the cells. Specifically, when the firstfluorescent dye labeling the BCR locus is irradiated with the light ofwavelength λ11, first fluorescence of wavelength λ21 is generated fromthe first fluorescent dye. When the second fluorescent dye labeling theABL locus is irradiated with the light of wavelength λ12, secondfluorescence of wavelength λ22 is generated from the second fluorescentdye. When the nuclear dye staining the nucleus is irradiated with thelight of wavelength λ13, third fluorescence of wavelength λ23 isgenerated from the nuclear dye. When the sample 10 flowing through theflow cell 110 is irradiated with the light of wavelength λ14, this lighttransmits through the cells. The transmitted light of wavelength λ14that has been transmitted through the cells is used for generating abright field image. For example, in the embodiment, the firstfluorescence is in a wavelength band of green light, the secondfluorescence is in a wavelength band of red light, and the thirdfluorescence is in a wavelength band of blue light.

The condenser lens 150 collects the first to third fluorescencegenerated from the sample 10 flowing through the flow channel 111 of theflow cell 110 and the transmitted light transmitted through the sample10 flowing through the flow channel 111 of the flow cell 110. Theoptical unit 151 has a configuration in which four dichroic mirrors arecombined. The four dichroic mirrors of the optical unit 151 reflect thefirst to third fluorescence and the transmitted light at slightlydifferent angles from each other and separate the light on a lightreceiving surface of the imaging unit 160. The condenser lens 152condenses the first to third fluorescence and the transmitted light.

The imaging unit 160 is constituted by a time delay integration (TDI)camera. The imaging unit 160 images the first to third fluorescence andthe transmitted light and outputs fluorescence images respectivelycorresponding to the first to third fluorescence and a bright fieldimage corresponding to the transmitted light as imaging signals to theimage processing device 200. The fluorescence images corresponding tothe first to third fluorescence are hereinafter respectively referred toas a “first image”, a “second image”, and a “third image”. The “firstimage”, “second image” and “third image” preferably have the same sizein order to analyze overlapping of bright spots. The “first image”,“second image”, and “third image” may be color images or gray scaleimages.

FIG. 2A shows examples of fluorescence images. In the first image ofFIG. 2A, a portion that looks like a dark dot shows a bright spot of thefirst fluorescence, that is, a target site labeled with the firstfluorescent dye. In the second image, although not as vivid as in thefirst image, a dark gray dot is observed in a light gray colorindicating a nucleus. This indicates a bright spot of the secondfluorescence, that is, a target site labeled with the second fluorescentdye. In the third image, a region of a substantially circular nucleus isrepresented in black. In a bright field image, the state of actual cellscan be observed. Each image in FIG. 2A is an image showing an example inwhich white blood cells after a pretreatment are placed on a glass slideand observed under a microscope, and, in raw data of the fluorescenceimage, brighter spots indicate higher fluorescence intensity and darkerspots indicate lower fluorescence intensity. In the first to thirdimages in FIG. 2A, the gradation of the imaged raw data is reversed andrepresented in gray scale. In the case where the sample 10 flowingthrough the flow cell 110 is imaged by the imaging unit 160 as describedabove, since the cells flow through the flow channel 111 in a state ofbeing separated from each other, the fluorescence image and the brightfield image are acquired for each cell.

Returning to FIG. 1, the image processing device 200 includes aprocessing unit 11, a storage unit 12, a display unit 13, and an inputunit 14 as a hardware configuration. The processing unit 11 isconstituted by a processor (central processing unit: CPU). The storageunit 12 is constituted by a readable and writable memory (random accessmemory: RAM) used as a work area for various processes of the processingunit 11, a read-only memory (ROM) for storing computer programs anddata, a hard disk, and the like. The processing unit 11 and the storageunit 12 can be configured by a general-purpose computer. The hard diskmay be included in the computer or may be placed as an external deviceof the computer. The display unit 13 is constituted by a display. Theinput unit 14 is constituted by a mouse, a keyboard, a touch paneldevice, or the like. The processing unit 11 transmits data to and fromthe storage unit 12 via a bus 15, and inputs and outputs data to andfrom the display unit 13, the input unit 14, and the measurement device100 via an interface 16.

The processing unit 11 reads out various computer programs stored in theROM or the hard disk to the RAM, executes the computer programs, andthus processes the fluorescence image of cells obtained by themeasurement of the sample 10 performed by the measurement device 100,and controls operations of the display unit 13, the input unit 14, andthe like. Specifically, the processing unit 11 extracts a plurality ofbright spots in a fluorescence image including a target site for eachcell, and changes the pixel value of each of the plurality of extractedbright spots according to the pixel value of the bright spot.

A “pixel value” in this specification refers to a digital value assignedto each pixel of an image, and in particular, in an output image(so-called raw image) from a camera, refers to a value of the luminanceof an imaging target object converted into a digital signal.

Hereinafter, an example of an image processing method of a fluorescenceimage performed by the processing unit 11 based on a computer programdefining a processing procedure for processing a fluorescence image of acell acquired by imaging the sample 10 will be described with referenceto FIG. 3. The computer program is stored in the storage unit 12 inadvance, but may be installed from a computer-readable portablerecording medium (not shown) such as a CD-ROM, or, for example, may beinstalled by being downloaded from an external server via a network (notshown).

As shown in FIG. 3, the processing unit 11 performs respective processesat an image acquisition step S1, an extraction step S2 of a bright spotand a nucleus region, a bright spot emphasis step S3, a pixel valueadjustment step S4, a pixel value changing step S5, an image combiningstep S6, and an image display step S7.

First, in step S1, the processing unit 11 acquires the first to thirdimages displayed in grayscale by gradation inversion of the raw datacaptured by the imaging unit 160. The processing unit 11 causes thestorage unit 12 to store the acquired first to third images.

Next, in step S2, the processing unit 11 extracts a bright spot (firstbright spot) of first fluorescence in the first image, a bright spot(second bright spot) of second fluorescence in the second image, and anucleus region in the third image.

More specifically, referring to FIGS. 2B to 2D, the third image shown atthe left end of FIG. 2B, the first image shown at the left end of FIG.2C, and the second image shown at the left end of FIG. 2D are acquiredfrom one cell flowing through the flow cell 110.

When a third image as shown at the left end of FIG. 2B is acquired, theprocessing unit 11 firstly generates a graph of pixel values and numberof pixels based on the pixel value of each pixel in horizontal (xdirection) m×vertical (y direction) n pixels constituting the thirdimage as shown in the center of FIG. 2B. The “number of pixels” of thevertical axis indicates the number of pixels. The number of pixels ofthe image is not particularly limited, is, for example, horizontal51×vertical 51. The spatial resolution per pixel is also notparticularly limited. Then, the processing unit 11 sets a thresholdvalue of the pixel value in the graph of FIG. 2B, and extracts a rangewhere pixels having pixel values larger than the threshold value as anucleus region as indicated by a broken line at the right end of FIG.2B.

Next, when a first image as shown at the left end of FIG. 2C isacquired, the processing unit 11 firstly generates a graph of pixelvalues and number of pixels based on the pixel value of each pixel inhorizontal (x direction) m×vertical (y direction) n pixels constitutingthe first image as shown in the center of FIG. 2C. Then, the processingunit 11 sets a threshold value of the pixel value in the graph of FIG.2C, for example, as a boundary between a bright spot and the backgroundbased on an Otsu method, and extracts a range where pixels having pixelvalues larger than the threshold value as a first bright spot asindicated by a broken line at the right end of FIG. 2C. When extractinga first bright spot from the first image, an extremely small bright spotand an extremely large bright spot are excluded. The size of a brightspot can be represented by the area of the bright spot in the firstimage (the number of pixels included in the bright spot). The positionof the nucleus region is compared with the position of the bright spotextracted from the first image, and a bright spot not included in thenucleus region is excluded. As a result, first bright spots areextracted from the first image, and the number and positions of thefirst bright spots are derived.

Next, when a second image as shown at the left end of FIG. 2D isacquired, the processing unit 11 generates a graph of pixel values andnumber of pixels based on the pixel value of each pixel in horizontal (xdirection) m×vertical (y direction) n pixels constituting the secondimage as shown in the center of FIG. 2D, similarly to the case of thefirst image. Then, the processing unit 11 sets a threshold value of thepixel value in the graph of FIG. 2D, and extracts a range where pixelshaving pixel values larger than the threshold value as a second brightspot as indicated by a broken line at the right end of FIG. 2D. Whenextracting a second bright spot from the second image, an extremelysmall bright spot and an extremely large bright spots are excluded. Thesize of a bright spot can be represented by the area of the bright spotin the second image (the number of pixels included in the bright spot).The position of the nucleus region is compared with the position of thebright spot extracted from the second image, and a bright spot notincluded in the nucleus region is excluded. As a result, second brightspots are extracted from the second image, and the number and positionsof the second bright spots are derived.

The positions of the nucleus region and the bright spots in each imagecan be measured by, for example, determining coordinate information (x,y) for horizontal (x direction) m×vertical (y direction) n pixelsconstituting each image and based on the coordinate information of aplurality of pixels included in the nucleus region and bright spots.

The processing unit 11 may extract a first bright spot, a second brightspot, and a nucleus region respectively from the first image, the secondimage, and the third image by calculation according to the aboveprocedure without generating the graphs as shown in the center of FIGS.2B to 2D. The extraction of the bright spots may be performed bydetermining the degree of matching between a distribution waveform ofnormal bright spots and a target region of determination and extractingthe target region of determination as a bright spot when the degree ofmatching is high. Although the processing unit 11 detects cells byextracting the nucleus region from the third image in the descriptionabove, the processing unit 11 may detect the cells based on a brightfield image. In the case where cells are detected based on the brightfield image, acquisition of the third image may be omitted. A brightspot in the present embodiment refers to a point of small fluorescencegenerated in the fluorescence image. More specifically, the bright spotrefers to the central point of a bright spot (the position of the pixelhaving the greatest pixel value (fluorescence intensity)). Theextraction of the bright spots can be performed by, for example,converting the color gradation of pixels other than the pixelsdesignated as bright spots to the same level as the background.

Returning to FIG. 3, next, in step S3, the processing unit 11 emphasizesbright spots by relatively increasing the pixel values of the extractedplurality of first bright spots and second bright spots respectively forthe first image and the second image with respect to the other region(background) than the bright spots.

First, a method of determining whether or not a cell is an abnormal cellwill be described.

FIG. 4A shows an arrangement example of bright spots of a normal cellhaving no chromosomal abnormality, that is, an example of bright spotpattern (negative pattern), and FIGS. 4B to 4D show examples of brightspot patterns (positive patterns) of abnormal cells. In any of FIGS. 4Ato 4D, each image is shown in a state of being superimposed with thethird image.

As shown in FIG. 4A, when there is no chromosomal abnormality such astranslocation of BCR locus and ABL locus, each gene exists as one pairin one nucleus, and each allele independently exists. Therefore, in thefirst image, there are two first bright spots in one nucleus region. Inthe second image, there are two second bright spots in one nucleusregion. In this case, if the first image and the second image imaged atthe same size are superimposed and combined, in the composite image, thetwo first bright spots and the two second bright spots are presentwithout overlapping in one nucleus region. Therefore, a cell in whichtwo first bright spots and two second bright spots are present in thenucleus region as shown in FIG. 4A is determined to be a normal cell inwhich no chromosomal abnormality is observed, that is, the chromosomeabnormality is negative.

In contrast, as shown in FIG. 4B, when a part of the ABL locus has movedto chromosome 9 due to translocation, there are two first bright spotsin the nucleus in the first image, and there are three second brightspots in the nucleus in the second image. In this case, when combiningthe first image and the second image, in the composite image, one firstbright spot, two second bright spots, and a bright spot (fused brightspot) of fourth fluorescence (yellow in the present embodiment) in whicha first bright spot and a second bright spot overlap each other arepresent in one nucleus. Therefore, as shown in FIG. 4B, a cell in whichthe respective bright spots are present is determined to be an abnormalcell in which translocation occurs in the BCR locus and the ABL locus,that is, chromosomal abnormality is positive.

As shown in FIG. 4C, when a part of the BCR locus has moved tochromosome 22 due to translocation and a part of the ABL locus has movedto the chromosome 9, there are three first bright spots in the nucleusin the first image, and there are three second bright spots in thenucleus in the second image. In this case, when combining the firstimage and the second image, in the composite image, one first brightspot, one second bright spots, and two fused bright spots in which firstbright spots and second bright spots overlap each other are present inone nucleus. Therefore, as shown in FIG. 4C, a cell in which therespective bright spots are present is determined to be an abnormal cellin which translocation occurs in the BCR locus and the ABL locus, thatis, chromosomal abnormality is positive.

As shown in FIG. 4D, when a part of the ABL locus has moved tochromosome 9 due to translocation, there are two first bright spots inthe nucleus in the first image, and there are two second bright spots inthe nucleus in the second image. In this case, when combining the firstimage and the second image, in the composite image, one first brightspot, one second bright spots, and one fused bright spot in which afirst bright spot and a second bright spot overlap each other arepresent in one nucleus. Therefore, as shown in FIG. 4D, a cell in whichthe respective bright spots are present is determined to be an abnormalcell in which translocation occurs in the BCR locus and the ABL locus,that is, chromosomal abnormality is positive.

In this manner, it is possible to determine whether or not each cell isan abnormal cell having a chromosomal abnormality based on the positionsand the number of the respective bright spots in the composite imageobtained by combining the first image and the second image. The firstbright spot, the second bright spot, and the fused bright spot can beindicated by color information in each image and composite image thereofsuch that the operator or the like can easily recognize the first brightspot, the second bright spot, and the fused bright spot by seeing thedisplay unit 13. That is, instead of displaying each image in grayscale, the color of each pixel of the first image can be displayed ingreen color gradation (RGB value) of the first fluorescence based on thepixel value, and thus a region bright in green (first fluorescence) canbe recognized as a first bright spot. In the second image, the color ofeach pixel can be displayed in red color gradation (RGB value) of thesecond fluorescence based on the pixel value, and thus a region brightin red (second fluorescence) can be recognized as a second bright spot.In a composite image in which the first image and the second image aresuperimposed, when there is a fused bright spot in which a first brightspot that is green (first fluorescence) and a second bright spot that isred (second fluorescence, a region bright in yellow (fourthfluorescence) can be recognized as a fused bright spot based on thecombination of the RGB values of pixels of the fused bright spot.Therefore, when the cell is an abnormal cell, a first bright spot thatis green (first fluorescence), a second bright spot that is red (secondfluorescence), a fused bright spot that is yellow (fourth fluorescence)are present in the nucleus region.

As shown in FIGS. 5A and 6A, respective bright spots extracted from thefirst image and the second image include a light bright spot and a darkbright spot. For example, in FIG. 5A, three first bright spots 2A to 2Care present in the first image. While the two first bright spots 2A and2B are light, the one first bright spot 2C is dark. Meanwhile, in FIG.6A, three second bright spots 3A to 3C are present in the second image.While the one second bright spot 3A is light, the two second brightspots 3B and 3C are dark. The lightness and darkness of a bright spotare indicated by the size (area) of the bright spot in FIGS. 5A and 6A.As shown in FIGS. 5A and 6A, the dark bright spots 2B, 2C and 3C have asmall difference in brightness from backgrounds 2D and 3D in respectiveimages, the dark bright spots 2B, 2C, and 3C are difficult for theoperator or the like to visually recognize, and thus the operator or thelike tends to miss the dark bright spots 2B, 2C, and 3C. Therefore, theprocessing unit 11 performs a bright spot emphasis process to clarifythe dark bright spots 2B, 2C, and 3C of weak fluorescence in the firstimage and the second image to make it easier to recognize dark brightspots in each image.

In the bright spot emphasis process, for example, as shown in FIGS. 5Band 6B, the pixel values of the first bright spots 2A to 2C and thesecond bright spots 3A to 3C can be relatively increased with respect tothe pixel values of the backgrounds 2D and 3D by decreasing the pixelvalues of pixels positioned in the backgrounds 2D and 3D of the firstimage and the second image to predetermined pixel values. The pixelvalues of the first bright spots 2A to 2C and the second bright spots 3Ato 3C can be relatively increased with respect to the pixel values ofthe backgrounds 2D and 3D by increasing the pixel values of pixelspositioned in the first bright spots 2A to 2C and the second brightspots 3A to 3C by multiplying the pixel values by predeterminedcoefficients. Further, the pixel values of the first bright spots 2A to2C and the second bright spots 3A to 3C can be relatively increased withrespect to the pixel values of the backgrounds 2D and 3D by increasingthe pixel values of pixels of the first image and the second image bymultiplying the pixels positioned in the first bright spots 2A to 2C andthe second bright spots 3A to 3C by larger coefficients or decreasingthe pixel values of pixels positioned in the backgrounds 2D and 3D bymultiplying the pixels positioned in the backgrounds 2D and 3D bysmaller coefficients.

The processing unit 11 causes the storage unit 12 to store the firstimage and the second image in which a first bright spot and a secondbright spot have been emphasized in the bright spot emphasis process.

Returning to FIG. 3, next, in step S4, the processing unit 11 adjuststhe pixel values of a bright spot extracted from one of the first imageand the second image based on pixel values of a bright spot extractedfrom the other of the first image and the second image. Specifically,when there is a difference between the pixel values of the first brightspot of the first image and the pixel values of the second bright spotof the second image, the highest values of the pixel values of bothbright spots are caused to match. For example, the highest values of thepixel values of both bright spots can be caused to match by increasingthe pixel values of pixels positioned in a bright spot of one imagewhose highest value of pixel values is lower by, for example,multiplying the pixel values by a predetermined coefficient. The highestvalues of the pixel values of both bright spots can be caused to matchby increasing the pixel values of pixels positioned in a bright spot ofthe image whose highest value of pixel values is lower by, for example,multiplying the pixel values by a predetermined first coefficient andincreasing the pixel values of pixels positioned in a bright spot of theimage whose highest value of pixel values is higher by, for example,multiplying the pixel values by a predetermined second coefficientsmaller than the first coefficient.

In the case where there is a large difference between the pixel valuesof a first bright spot of green (first fluorescence) and the pixelvalues of a second bright spot of red (second fluorescence) at a fusedbright spot where the first bright spot and the second bright spot arefused, the fused bright spot may not be displayed yellow (fourthfluorescence). For example, if the pixel values of the first bright spotof green (first fluorescence) is significantly larger than the pixelvalue of the second bright spot of red (second fluorescence), the colorof the fused bright spot becomes close to green (first fluorescence) andbecomes less likely to appear yellow (fourth fluorescence). In thiscase, even if the operator or the like visually recognizes the fusedbright spot, the operator or the like does not recognize the spot as afused bright spot but mistakenly recognizes the spot as a first brightspot. Therefore, the processing unit 11 corrects the pixel values of thefirst bright spot of the first image and the pixel values of the secondbright spot of the second image to the same level by the adjustmentprocess of the pixel values to cause the fused bright spot in which thefirst bright spot of green (first fluorescence) and the second brightspot of red (second fluorescence) are fused is displayed yellow (fourthfluorescence) such that it is easier to recognize the fused bright spot.

The processing unit 11 causes the storage unit 12 to store the firstimage and the second image in which pixel values of a first bright spotand a second bright spot have been adjusted in the adjustment process ofpixel values.

Returning to FIG. 3, next, in step S5, the processing unit 11 changes,according to the pixel values of each bright spot, the pixel values of aplurality of first bright spots and a plurality of second bright spotsrespectively extracted from the first image and the second image.

As shown in FIG. 5A and FIG. 6A, among the bright spots 2A to 2C and 3Ato 3C extracted from the first image and the second image, the darkbright spots 2B, 2C, and 3C are difficult for an observer to visuallyrecognize in the respective images, and are likely to be missed by theobserver. As shown in FIGS. 5B and 6B, the dark bright spots 2B, 2C, and3C are harder for the observer to visually recognize than the lightbright spots 2A, 2B, 3A, and 3B in the respective images even after theeach image after the bright spot emphasis process. Besides, in the casewhere there is large difference between the pixel values of the darkbright spots 2B, 2C, and 3C and the light bright spots 2A, 3A, and 3B,and, for example, the bright spot 3B of dark red (second fluorescence)of the second image overlaps with the bright spot 2B of light green(first fluorescence) of the first image to form a fused bright spot whencombining the first image and the second image, the color of the fusedbright spot 4B becomes close to the green (first fluorescence) of thelight first bright spot and becomes less likely to appear yellow. Forthis reason, an operator or the like is likely not to recognize the spotas a fused bright spot and mistakenly recognize the spot as anotherbright spot.

Therefore, the processing unit 11 changes the pixel values of aplurality of bright spots having different pixel values at differentchange rates in each of the first image and the second image by theprocess of changing the pixel values. The change rates include increaserates and decrease rates. For example, as shown in FIGS. 5C and 6C, thepixel value of a pixel having a smaller pixel value is increased with ahigher increase rate. As a result, regarding the dark bright spots 2C,3B, and 3C having small pixel values, the pixel values thereof areincreased more greatly than the light bright spots 2A, 2B, 3A, and 3Bhaving large pixel values so as to brighten the dark bright spots 2C,3B, and 3C to facilitate recognizing the dark bright spots 2C, 3B, and3C. FIG. 8 shows a composite image in which the first image, the secondimage, and the third image are combined. As can be seen by comparingFIG. 8A with FIG. 8B, the dark bright spots 2C, 3B, and 3C having smallpixel values are made easier to recognize without being buried in thenucleus region 5 of the back ground in the case where the bright spots2A to 2C and 3A to 3C of the first image and the second image aresuperimposed on the nucleus region 5 of the third image. Besides, as canbe seen from the comparison between FIG. 7B and FIG. 8B and thecomparison between FIG. 7A and FIG. 8A, the processing unit 11 reducesthe difference in pixel value between the dark bright spots 2C, 3B, and3C and the light bright spots 2A, 2B, and 3A having large pixel valuessuch that the fused bright spot 4B in which the first bright spot 2B ofgreen (first fluorescence) and the second bright spot 3B of red (secondfluorescence) are fused is displayed in a color closer to yellow (fourthfluorescence) and that it is easier to recognize the fused bright spot4B when the first image and the second image are combined.

In the process of changing pixel values, the pixel values may be changedin any method as long as the method follows a rule that the pixel valuesof pixels included in each bright spot in each image is increased byusing a larger increase rate for a pixel having a smaller pixel value,and the method of changing the pixel values is not particularly limited.In the process of changing pixel values, it is preferable to increasethe pixel values by using a larger increase rate for a pixel having asmaller pixel value while maintaining the magnitude relationship betweenthe pixel values of respective pixels included in each bright spot. Itis preferable to maintain the pixel value of a pixel having the highestpixel value among the pixels whose pixel values are to be changed andwhich are included in each bright spot. This makes it possible toreproduce the original brightness relationship between the bright spotsin each image after the changing process.

As the process of changing pixel values, for example, as shown in FIG.9A, it is possible to change an unchanged pixel value x to a changedpixel value y by Expression 1 below for each pixel included in an imageto be changed. In Expression 1 and FIG. 9A, both the highest pixelvalues of the unchanged pixel value x and the changed pixel value y area highest pixel value M of a pixel before being changed, and a range ofpixel values 0 to M is normalized by the highest pixel value M.According to Expression 1 and FIG. 9A, for a pixel whose unchanged pixelvalue x is smaller than a threshold value Th, the changed pixel value ybecomes equal to the unchanged pixel value x, and thus the pixel valueis maintained without being changed. In contrast, for a pixel whoseunchanged pixel value x is larger than the threshold value Th, the pixelvalue of a pixel having a smaller pixel value is increased with a largerincrease rate by a predetermined functional formula while maintainingthe magnitude relationship between pixel values. By setting thethreshold value Th to the lowest pixel value of pixel values of pixelsincluded in each bright spot, just the pixel values of each bright spotcan be changed according to the pixel values thereof.y=x^(γ) (where x≥Th and γ<1) and y=x (where x<Th)  [Expression 1]

In another example of the process of changing pixel values, as shown inFIG. 9B, it is possible to change the unchanged pixel value x to thechanged pixel value y by Expression 2 below for each pixel included inthe image to be changed. Also in Expression 2 below and FIG. 9B, boththe highest pixel values of the unchanged pixel value x and the changedpixel value y are the highest pixel value M of a pixel before beingchanged, and the range of pixel values 0 to M is normalized by thehighest pixel value M. Also according to Expression 2 and FIG. 9B, for apixel whose unchanged pixel value x is smaller than the threshold valueTh, the changed pixel value y becomes equal to the unchanged pixel valuex, and thus the pixel value is maintained without being changed. Incontrast, for a pixel whose unchanged pixel value x is larger than thethreshold value Th, the pixel value of a pixel having a smaller pixelvalue is increased with a larger increase rate by a predeterminedfunctional formula while maintaining the magnitude relationship betweenpixel values. By setting the threshold value Th to the lowest pixelvalue of pixel values of pixels included in each bright spot, just thepixel values of each bright spot can be changed according to the pixelvalues thereof.y=(1−b)x+b (where x≥Th), and y=x (where x<Th)  [Expression 2]

In the process of changing pixel values, the pixel values may be changedby a reference process using a predetermined look up table following therule described above instead of arithmetic processing using a functionalexpression.

The processing unit 11 causes the storage unit 12 to store the firstimage and the second image in which pixel values of a first bright spotand a second bright spot have been changed in the process of changingpixel values.

Returning to FIG. 3, next, in step S6, the processing unit 11 combinesthe first image and the second image, from which the bright spotsdescribed above have been extracted and which have been subjected to theimage processing, and the third image from which the nucleus region hasbeen extracted. The processing unit 11 is capable of generating, byimage combination, a plurality of kinds of composite images such as acomposite image of the first image and the third image, a compositeimage of the second image and the third image, a composite image of thefirst image and the second image, and a composite image of the firstimage, the second image, and the third image. It is not always necessaryto create all of the above-described plurality of kinds of compositeimages. The processing unit 11 causes the storage unit 12 to store eachcomposite image that has been generated.

Finally, in step S7, the processing unit 11 displays the first image andthe second image, from which the bright spots described above have beenextracted and which have been subjected to the image processing, thethird image from which the nucleus region has been extracted, andfurther each composite image generated by image combination on thedisplay unit 13. The processing unit 11 does not necessarily display allof the above-described images on the display unit 13, and can displayingonly necessary images selected by the operator or the like on thedisplay unit 13.

The operator or the like observes each image displayed on the displayunit 13 and checks whether or not each cell is an abnormal cell based onthe color and number of bright spots in each image.

According to the present disclosure, when the operator or the likeobserves a fluorescence image of a cell displayed on the display unit13, first, pixel values of pixels in each bright spot is relativelyenhanced with respect to pixel values of pixels in the backgroundoutside the bright spot by the bright spot emphasis process in the firstimage and the second image from which bright spots have been extracted.Therefore, even if dark bright spots having low pixel values areextracted from each image, it is easier for the operator or the like tovisually recognize the dark bright spots. In the first image and thesecond image, dark bright spots having low pixel values are enhanced bythe process of changing pixel values such that the difference betweenthe pixel values of the dark bright spots and the pixel values of lightbright spots having high pixel values is reduced, and thus it is furthereasier for the operator or the like to visually recognize the darkbright spots. In addition, since dark bright spots having low pixelvalues are enhanced by the process of changing pixel values such thatthe difference between the pixel values of the dark bright spots and thepixel values of light bright spots having high pixel values is reducedin the first image and the second image, a first bright spot and asecond bright spot are superimposed in a fused bright spot in acomposite image of the first image and the second image in a state inwhich the difference between pixel values thereof is reduced.Accordingly, the fused bright spot is displayed in yellow of fourthfluorescence or a color close to yellow, and thus it is easier for theoperator or the like to detect the fused bright spot by visualobservation. The fused bright spot in the composite image of the firstimage and the second image can be displayed yellow of fourthfluorescence by the adjustment process of pixel values between the firstbright spot of the first image and the second bright spot of the secondimage, and thus it is further easier for the operator or the like todetect the fused bright point by visual observation.

As described above, according to the present disclosure, even in thecase where a light bright spot having high pixel values and a darkbright spot having low pixel values are present in one fluorescenceimage, confirmation of a bright spot in image observation and detectionof a fused bright spot in analysis of a composite image of twofluorescence images can be performed easily. Therefore, it is possibleto easily and highly accurately determine whether each cell is a normalcell or an abnormal cell.

Although one embodiment of the present disclosure has been describedabove, the present disclosure is not limited to the present embodimentdescribed above, and various modifications are possible withoutdeparting from the gist of the present disclosure.

For example, in the present embodiment described above, the processingunit 11 increases the pixel values of each bright spot in eachfluorescence image (first image and second image) by using a largerincrease rate for a pixel having a smaller pixel value in the process ofchanging the pixel values in step S5 of FIG. 3. Thus, the differencebetween the pixel values of a dark bright spot and a light bright spotin each fluorescence image is reduced. However, the method of theprocess of changing pixel values is not limited to this. For example,the difference between the pixel values of a dark bright spot and alight bright spot in each fluorescence image can be reduced bydecreasing the pixel value of a pixel having a larger pixel value with alarger decrease rate in each bright spot in each fluorescence image.

In the present embodiment described above, regarding each fluorescenceimage (first image and second image) captured by the imaging unit 160,the processing unit 11 displays, on the display unit 13, fluorescenceimages after image processing. However, among fluorescence images beforeand after the image processing, any fluorescence image selected by theinput unit 14 may be displayed on the display unit 13. That is, thefirst image, the second image, and the third image before the imageprocessing by the processing unit 11, and a composite image in which atleast two of these are combined may be displayed on the display unit 13.

The processing unit 11 performs, in the processing procedure ofprocessing a fluorescence image of FIG. 3, the adjustment process ofpixel values of bright spots between a plurality of fluorescence imagesin step S4 and the process of changing pixel values of a plurality ofbright spots in each fluorescence image in step S5. However, theadjustment process of pixel values of step S4 may be performed after theprocess of changing pixel values of step S5. The processing unit 11 doesnot need to perform and may omit the adjustment process of pixel valuesof step S4.

The processing unit 11 performs, in the processing procedure ofprocessing a fluorescence image of FIG. 3, the extraction process of abright spot from a fluorescence image in step S2, and then the brightspot emphasis process in the fluorescence image in step S3. However, thebright spot emphasis process of step S3 may be performed after theadjustment process of pixel values of step S4 or after the process ofchanging pixel values of step S5. The processing unit 11 does not needto perform and may omit the bright spot emphasis process of step S3.

In the above-described fluorescence image analyzing apparatus 1 of thepresent embodiment, the measurement device 100 shown in FIG. 1 may bereplaced by a measurement device 400 including a fluorescence microscopeshown in FIG. 10.

The measurement device 400 shown in FIG. 10 includes light sources 410to 412, a mirror 420, dichroic mirrors 421 and 422, a shutter 430, aquarter-wave plate 431, a beam expander 432, a condenser lens 433, adichroic mirror 434, an objective lens 435, a stage 440, a condenserlens 450, an imaging unit 451, and controllers 460 and 461. On the stage440, a glass slide 441 is installed. On the glass slide 441, the sample10 (shown in FIG. 1) prepared by the pretreatment in the pretreatmentdevice 300 is placed.

The light sources 410 to 412 are respectively similar to the lightsources 120 to 122 shown in FIG. 1. The mirror 420 reflects light fromthe light source 410. The dichroic mirror 421 transmits light from thelight source 410 and reflects light from the light source 411. Thedichroic mirror 422 transmits light from the light sources 410 and 411and reflects light from the light source 412. The optical axes of thelight from the light sources 410 to 412 are matched with each other bythe mirror 420 and the dichroic mirrors 421 and 422.

The shutter 430 is driven by the controller 460 to switch between astate of transmitting light emitted from the light sources 410 to 412and a state of blocking light emitted from the light sources 410 to 412.As a result of this, the irradiation time of the sample 10 with light isadjusted. The quarter-wave plate 431 converts linearly polarized lightemitted from the light sources 410 to 412 into circularly polarizedlight. Fluorescent dye bound to a probe reacts to light of apredetermined polarization direction. Therefore, by convertingexcitation light emitted from the light sources 410 to 412 intocircularly polarized light, the polarization direction of the excitationlight becomes more likely to match the polarization direction to whichthe fluorescent dye reacts. This makes it possible to efficiently excitefluorescence in the fluorescent dye. The beam expander 432 expands alight irradiation area on the glass slide 441. The condenser lens 433collects light so that the glass slide 441 is irradiated with parallellight from the objective lens 435.

The dichroic mirror 434 reflects light emitted from the light sources410 to 412, and transmits fluorescence generated from the sample 10. Theobjective lens 435 guides the light reflected by the dichroic mirror 434to the glass slide 441. The stage 440 is driven by the controller 461.The fluorescence generated from the sample 10 passes through theobjective lens 435 and passes through the dichroic mirror 434. Thecondenser lens 450 collects the fluorescence transmitted through thedichroic mirror 434 and guides the light to an imaging surface 452 ofthe imaging unit 451. The imaging unit 451 captures an image of thefluorescence radiated on the imaging surface 452, and generates afluorescence image. The imaging unit 451 is constituted by, for example,a charge coupled device (CCD).

The controllers 460 and 461 and the imaging unit 451 are connected tothe processing unit 11 shown in FIG. 1, and the processing unit 11controls the controllers 460 and 461 and the imaging unit 451 andreceives the fluorescence image captured by the imaging unit 451. Unlikethe case where the flow cell 110 is used as shown in FIG. 1, thefluorescence image captured by the imaging unit 451 may be in a state inwhich cells are in close contact with each other as shown in FIG. 2A.Therefore, the processing unit 11 performs a process of dividing theacquired fluorescence image for each nucleus of a cell, a process ofsetting a region corresponding to one nucleus of a cell in thefluorescence image, or the like.

Also in the measurement device 400 shown in FIG. 10, three fluorescenceimages (first image to third image) can be acquired as in the presentembodiment. Therefore, by generating a composite image by imageprocessing of the fluorescence images, the operator or the like caneasily and highly accurately determine whether each cell is a normalcell or an abnormal cell.

In the fluorescence image analyzing apparatus 1 of the presentembodiment described above, the processing unit 11 may be connected tothe pretreatment device 300 via the interface 16 so that data can beinputted and outputted therebetween.

A storage medium storing a computer program defining a processingprocedure for processing the fluorescence images of cells by theprocessing unit 11 of the image processing device 200 described abovecan also be provided.

In the fluorescence image analyzing apparatus 1 of the presentembodiment described above, a BCR/ABL fusion gene. In addition to theBCR/ABL fusion gene, examples of chromosomal translocations for which afused bright spot can be detected by the FISH method include AML1/ETO(MTG8) fusion gene (t(8; 21)), PML/RARα fusion gene (t(15; 17)), AML1(21q22) translocation, MLL (11q23) translocation, TEL (12p13)translocation, TEL/AML1 fusion gene (t(12; 21)), IgH (14q32)translocation, CCND1 (BCL1)/IgH fusion gene (t(11; 14)), BCL2 (18q21)translocation, IgH/MAF fusion gene (t(14; 16)), IgH/BCL2 fusion gene(t(14; 18)), c-myc/IgH fusion gene (t(8; 14)), FGFR3/IgH fusion gene(t(4; 14)), BCL6 (3q27) translocation, c-myc (8q24) translocation, MALT1(18q21) translocation, API2/MALT1 fusion gene (t(11; 18) translocation),TCF3/PBX1 fusion gene (t(1; 19) translocation), EWSR1 (22q12)translocation, and PDGFRβ (5q32) translocation.

Another exemplary embodiment can be applied to chromosomal abnormalityof the ALK locus. In a positive pattern, since the ALK gene is cleaved,only one fused bright spot is recognized (in the case where only one ofthe alleles is cleaved) or no fused bright spot is recognized (in thecase where both of the alleles are cleaved). The negative pattern andthe positive pattern are the same for the ROS1 gene and the RET gene inaddition to the ALK gene.

Another exemplary embodiment can be applied to chromosomal abnormalityof deletion of the long arm of chromosome 5 (5q). For example, the firstfluorescently labeled probe is designed to bind to the long arm ofchromosome 5, and the second fluorescently labeled probe is designed tobind to the centromere of chromosome 5. In the negative pattern, sincethe number of centromere of chromosome 5 is the same as the number oflong arm of chromosome 5, the number of bright spots (first brightspots) of the first fluorescently labeled probe and bright spots (secondbright spots) of the second fluorescently labeled probe are each two,reflecting the number of homologous chromosomes. In the positivepattern, long arm deletion occurs in one or both of chromosome 5 and thenumber of the first bright spots is only one or zero. This negativepattern and positive pattern are the same for deletion of short arm orlong arm of other chromosomes. Examples of long arm deletion of otherchromosomes include long arm deletion of chromosome 7 and chromosome 20.Other examples showing similar positive patterns and negative patternsinclude 7q31 (deletion), p16 (9p21 deletion analysis), IRF-1 (5q31)deletion, D20S108 (20q12) deletion, D13S319 (13q14) deletion, 4q12deletion, ATM (11q22.3) deletion, and p53 (17p13.1) deletion.

Another exemplary embodiment can be applied to trisomy of chromosome 8.The first fluorescently labeled probe binds to, for example, thecentromere of chromosome 8. In the positive pattern, there are threefirst bright spots. In the negative pattern, there are two first brightspots. Such a bright spot pattern is the same for trisomy of chromosome12. In chromosome 7 monosomy, for example, in the case of using thefirst fluorescently labeled probe that binds to the centromere ofchromosome 7, the positive pattern has one first bright spot. In thenegative pattern, there are two first bright spots.

What is claimed is:
 1. A fluorescence image analyzing apparatuscomprising: a light source that emits light to a sample including a celllabeled with a fluorescent dye at a target site; an imaging unit thatcaptures a fluorescence image of the cell that emit fluorescence bybeing irradiated with the light; a processing unit that processes thefluorescence image captured by the imaging unit; and a display unit thatdisplays the fluorescence image processed by the processing unit,wherein the processing unit performs: an extraction process ofextracting one or more bright spots in the fluorescence image includingthe target site, a changing process of changing a pixel value of atleast one of extracted bright spots based on the pixel value of eachbright spot, and a display process of displaying the fluorescence imagewhose pixel value is changed on the display unit.
 2. The fluorescenceimage analyzing apparatus according to claim 1, wherein the imaging unitcaptures a first fluorescence image including a target site labeled witha first fluorescent label and a second fluorescence image including atarget site labeled with a second fluorescent label, wherein, in theextraction process, the processing unit extracts one or more brightspots in the first fluorescence image and one or more bright spots inthe second fluorescence image for each cell, wherein, in the changingprocess, the processing unit changes a pixel value of at least one ofbright spots extracted from the first fluorescence image and the secondfluorescence image, based on the pixel value of each bright spot, andwherein the processing unit further performs an image combining processof combining the first fluorescence image and the second fluorescenceimage whose pixel value is changed.
 3. The fluorescence image analyzingapparatus according to claim 2, wherein the target site of the firstfluorescence image is a BCR locus and the target site of the secondfluorescence image is an ABL locus.
 4. The fluorescence image analyzingapparatus according to claim 2, wherein the processing unit furtherperforms an adjustment process of adjusting a pixel value of a brightspot extracted from one of the first fluorescence image and the secondfluorescence image based on a pixel value of a bright spot extractedfrom another of the first fluorescence image and the second fluorescenceimage.
 5. The fluorescence image analyzing apparatus according to claim1, wherein, in the changing process, the processing unit changes pixelvalues of a plurality of bright spots having different pixel valuesextracted from a fluorescence image, by respectively using change ratesdifferent from one another.
 6. The fluorescence image analyzingapparatus according to claim 5, wherein, in the changing process, theprocessing unit increases a pixel value of a pixel having a smallerpixel value by using a larger increase rate.
 7. The fluorescence imageanalyzing apparatus according to claim 5, wherein, in the changingprocess, the processing unit decreases a pixel value of a pixel having alarger pixel value by using a larger decrease rate.
 8. The fluorescenceimage analyzing apparatus according to claim 1, wherein, in the changingprocess, the processing unit changes pixel values of bright spots whilemaintaining a magnitude relationship between pixel values of pixels. 9.The fluorescence image analyzing apparatus according to claim 1, whereinthe processing unit further performs an emphasis process of relativelyincreasing pixel values of the plurality of extracted bright spots withrespect to a pixel value of a region outside the plurality of extractedbright spots.
 10. The fluorescence image analyzing apparatus accordingto claim 9, wherein, in the emphasis process, the processing unitdecreases the pixel value of the region outside the plurality ofextracted bright spots.
 11. The fluorescence image analyzing apparatusaccording to claim 1, wherein the imaging unit captures a fluorescenceimage including a nucleus, and wherein, in the extraction process, theprocessing unit further extracts, for each cell, a nucleus region in thefluorescence image including the nucleus.
 12. The fluorescence imageanalyzing apparatus according to claim 11, wherein the processing unitfurther performs an image combining process of combining thefluorescence image including the nucleus and the fluorescence imageincluding the target site.
 13. The fluorescence image analyzingapparatus according to claim 1, further comprising a flow cell throughwhich the sample flows, wherein the light source irradiates the sampleflowing through the flow cell with light.
 14. The fluorescence imageanalyzing apparatus according to claim 1, further comprising an inputunit, wherein, regarding the fluorescence image captured by the imagingunit, the processing unit displays, on the display unit, a fluorescenceimage selected by the input unit from a fluorescence image before imageprocessing and a fluorescence image after image processing.
 15. An imageprocessing method of a fluorescence image in which a fluorescence imageof a cell acquired by measuring a sample subjected to a pretreatment forlabeling a target site in the cell with a fluorescent dye is processed,the image processing method comprising the steps of: extracting one ormore bright spots in the fluorescence image including the target site;and changing a pixel value of at least one of extracted bright spotsbased on the pixel value of each bright spot.
 16. The image processingmethod according to claim 15, wherein a first fluorescence imageincluding a target site labeled with a first fluorescent label and asecond fluorescence image including a target site labeled with a secondfluorescent label are captured.
 17. The image processing methodaccording to claim 15, wherein, in the changing process, pixel values ofa plurality of bright spots having different pixel values extracted froma fluorescence image, by respectively using change rates different fromone another.
 18. The image processing method according to claim 15,wherein, a fluorescence image including a nucleus is captured, and inthe extraction process, a nucleus region in the fluorescence imageincluding the nucleus is extracted.
 19. The image processing methodaccording to claim 15, further comprising: irradiating the sampleflowing through a flow cell with light.
 20. A non-transitory tangiblemedia storing a computer program for causing a computer to execute imageprocessing of a fluorescence image of a cell acquired by measuring asample subjected to a pretreatment for labeling a target site in thecell with a fluorescent dye, the image processing including: anextraction process of extracting, for each cell, one or more brightspots in the fluorescence image including the target site; and achanging process of changing a pixel value of at least one of extractedbright spots based on the pixel value of each bright spot.